infectious serum or culture-grown virus?

Schmallenberg virus (SBV), discovered in Europe in 2011, causes mild transient disease in adult ruminants, but fetal infection can lead to severe malformation in cattle, sheep and goats.To elucidate the pathogenesis of this novel orthobunyavirus, considerable efforts are required. A reliable and standardized infection model is essential for in vivo studies. In the present study, two groups of four cattle were inoculated with either serum passaged in cattle only or cell culture-grown virus. The replication of culture-grown SBV in cattle was reduced compared to virus inoculated via infectious serum. In a second experiment, the infectious serum was titrated in calves; the tested batch contained 102.83 infectious doses per mL. Hence, serum-borne virus that was only passaged in the natural host is a suitable option for a standardized SBV infection model

Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE: Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines

Full Genome Characterisation of Bluetongue Virus Serotype 6 from the Netherlands 2008 and Comparison to Other Field and Vaccine Strains

In mid September 2008, clinical signs of bluetongue (particularly coronitis) were observed in cows on three different farms in eastern Netherlands (Luttenberg, Heeten, and Barchem), two of which had been vaccinated with an inactivated BTV-8 vaccine (during May-June 2008). Bluetongue virus (BTV) infection was also detected on a fourth farm (Oldenzaal) in the same area while testing for export. BTV RNA was subsequently identified by real time RT-PCR targeting genome-segment (Seg-) 10, in blood samples from each farm. The virus was isolated from the Heeten sample (IAH “dsRNA virus reference collection” [dsRNA-VRC] isolate number NET2008/05) and typed as BTV-6 by RT-PCR targeting Seg-2. Sequencing confirmed the virus type, showing an identical Seg-2 sequence to that of the South African BTV-6 live-vaccine-strain. Although most of the other genome segments also showed very high levels of identity to the BTV-6 vaccine (99.7 to 100%), Seg-10 showed greatest identity (98.4%) to the BTV-2 vaccine (RSAvvv2/02), indicating that NET2008/05 had acquired a different Seg-10 by reassortment. Although Seg-7 from NET2008/05 was also most closely related to the BTV-6 vaccine (99.7/100% nt/aa identity), the Seg-7 sequence derived from the blood sample of the same animal (NET2008/06) was identical to that of the Netherlands BTV-8 (NET2006/04 and NET2007/01). This indicates that the blood contained two different Seg-7 sequences, one of which (from the BTV-6 vaccine) was selected during virus isolation in cell-culture. The predominance of the BTV-8 Seg-7 in the blood sample suggests that the virus was in the process of reassorting with the northern field strain of BTV-8. Two genome segments of the virus showed significant differences from the BTV-6 vaccine, indicating that they had been acquired by reassortment event with BTV-8, and another unknown parental-strain. However, the route by which BTV-6 and BTV-8 entered northern Europe was not established

An Equine Herpesvirus Type 1 (EHV-1) Expressing VP2 and VP5 of Serotype 8 Bluetongue Virus (BTV-8) Induces Protection in a Murine Infection Model

Bluetongue virus (BTV) can infect most species of domestic and wild ruminants causing substantial morbidity and mortality and, consequently, high economic losses. In 2006, an epizootic of BTV serotype 8 (BTV-8) started in northern Europe that caused significant disease in cattle and sheep before comprehensive vaccination was introduced two years later. Here, we evaluate the potential of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus, as a novel vectored DIVA (differentiating infected from vaccinated animals) vaccine expressing VP2 of BTV-8 alone or in combination with VP5. The EHV-1 recombinant viruses stably expressed the transgenes and grew with kinetics that were identical to those of parental virus in vitro. After immunization of mice, a BTV-8-specific neutralizing antibody response was elicited. In a challenge experiment using a lethal dose of BTV-8, 100% of interferon-receptor-deficient (IFNAR−/−) mice vaccinated with the recombinant EHV-1 carrying both VP2 and VP5, but not VP2 alone, survived. VP7 was not included in the vectored vaccines and was successfully used as a DIVA marker. In summary, we show that EHV-1 expressing BTV-8 VP2 and VP5 is capable of eliciting a protective immune response that is distinguishable from that after infection and as such may be an alternative for BTV vaccination strategies in which DIVA compatibility is of importance

Proof of Proficiency of Decentralized Foot-and-Mouth Disease Virus Diagnostics in Germany

A proficiency test was performed to verify that the regional veterinary laboratories in Germany can provide reliable foot-and-mouth disease virus (FMDV) diagnostics. Overall, 24 samples were to be analyzed for FMDV-specific nucleic acids by real-time RT-PCR, and 16 samples had to be tested by ELISA for antibodies against non-structural proteins of FMDV. For both methods, a range of dilutions of the original materials (inactivated FMDV vaccine or convalescent serum from infected animals, respectively) was prepared, and negative samples were included as well. All 23 participating laboratories were able to detect FMDV genome down to a dilution of 1:100,000 of the vaccine preparation. Even at a dilution of 1:1,000,000, FMDV genome was detected by more than half of the participants. With the antibody ELISA, all sera were correctly identified by all participating laboratories. No false-positive results were returned with either method. All participating laboratories were found to be fully proficient in FMDV diagnostics

Pathogenicity study in sheep using reverse-genetics-based reassortant bluetongue viruses.

Bluetongue (BT) disease, caused by the non-enveloped bluetongue virus (BTV) belonging to the Reoviridae family, is an economically important disease that affects a wide range of wild and domestic ruminants. Currently, 26 different serotypes of BTV are recognized in the world, of which BTV-8 has been found to exhibit one of the most virulent manifestations of BT disease in livestock. In recent years incursions of BTV-8 in Europe have resulted in significant morbidity and mortality not only in sheep but also in cattle. The molecular and genetic basis of BTV-8 pathogenesis is not known. To understand the genetic basis of BTV-8 pathogenicity, we generated reassortant viruses by replacing the 3 most variable genes, S2, S6 and S10 of a recent isolate of BTV-8, in different combinations into the backbone of an attenuated strain of BTV-1. The growth profiles of these reassortant viruses were then analyzed in two different ovine cell lines derived from different organs, kidney and thymus. Distinct patterns for each reassortant virus in these two cell lines were observed. To determine the pathogenicity of these reassortant viruses, groups of BTV-susceptible sheep were infected with each of these viruses. The data suggested that the clinical manifestations of these two different serotypes, BTV-1 and BTV-8, were slightly distinct and BTV-1, when comprising all 3 genome segments of BTV-8, behaved differently to BTV-1. Our results also suggested that the molecular basis of BT disease is highly complex

After nasopharyngeal infection, foot-and mouth disease virus serotype A RNA is shed in bovine milk without associated mastitis

Foot-and-mouth disease (FMD) is a highly contagious aphthoviral infection of cloven-hoofed animals, inducing vesiculopustular stomatitis, pododermatitis, and thelitis. Vesicular fluid represents a major pathway of virus excretion, but bovine milk is another important source of virus shedding. We describe here the time course of FMD virus (FMDV) excretion in the milk and characterize associated lesions in the mammary gland. Three dairy cows were infected by nasopharyngeal instillation of FMDV and monitored over 12d. Autopsy was performed at the end of the study, and specimens were collected for histopathology, IHC, and RT-qPCR. All 3 cows developed fever, drooling, vesiculopustular stomatitis, interdigital dermatitis, and thelitis. FMDV RNA was detectable in whole milk until the end of the trial, but only transiently in saliva, nasal secretions, and blood serum. Although histology confirmed vesiculopustular lesions in the oral and epidermal specimens, the mammary glands did not have unequivocal evidence of FMDV-induced inflammation. FMDV antigen was detectable in skin and oral mucosa, but not in the mammary gland, and FMDV RNA was detectable in 9 of 29 samples of squamous epithelia but only in 1 of 12 samples of mammary gland

Real-Time Quantitative Reverse Transcription-PCR Assays Specifically Detecting Bluetongue Virus Serotypes 1, 6, and 8▿ †

Bluetongue virus (BTV) is a major pathogen of ruminants. Especially serotypes 1, 6, and 8 are of concern to veterinary authorities in central Europe. This article describes highly sensitive real-time reverse transcription-PCR assays directed to BTV genome segment 2 for specific detection of BTV-1, -6, or -8 in animal samples